Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The Mad2 spindle checkpoint protein has two distinct natively folded states

Nature Structural & Molecular Biology volume 11, pages 338–345 (2004)Cite this article

Abstract

The spindle checkpoint delays chromosome segregation in response to misaligned sister chromatids during mitosis, thus ensuring the fidelity of chromosome inheritance. Through binding to Cdc20, the Mad2 spindle checkpoint protein inhibits the target of this checkpoint, the ubiquitin protein ligase APC/CCdc20. We now show that without cofactor binding or covalent modification Mad2 adopts two distinct folded conformations at equilibrium (termed N1-Mad2 and N2-Mad2). The structure of N2-Mad2 has been determined by NMR spectroscopy. N2-Mad2 is much more potent in APC/C inhibition. Overexpression of a Mad2 mutant that specifically sequesters N2-Mad2 partially blocks checkpoint signaling in living cells. The two Mad2 conformers interconvert slowly in vitro, but interconversion is accelerated by a fragment of Mad1, an upstream regulator of Mad2. Our results suggest that the unusual two-state behavior of Mad2 is critical for spindle checkpoint signaling.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Mad2 R133A has two natively folded monomeric conformers.
Figure 2: Ribbon drawing of the structures of N1-Mad2 (left)16, N2-Mad2 R133A (middle, this study), and ligand-bound Mad2 (right)11.
Figure 3: N2-Mad2 more potently blocks the activity of APC/C than does N1-Mad2.
Figure 4: Endogenous Mad2 protein from HeLa cells exhibits the N1 conformation.
Figure 6: Mad1 facilitates the N1-N2 conversion of Mad2 in vitro.
Figure 5: Overexpression of ΔC-Mad2 partially blocks spindle checkpoint signaling in living cells.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Nasmyth, K. Segregating sister genomes: the molecular biology of chromosome separation. Science 297, 559–565 (2002).

    Article  CAS  Google Scholar 

  2. Cleveland, D.W., Mao, Y. & Sullivan, K.F. Centromeres and kinetochores. From epigenetics to mitotic checkpoint signaling. Cell 112, 407–421 (2003).

    Article  CAS  Google Scholar 

  3. Yu, H. Regulation of APC-Cdc20 by the spindle checkpoint. Curr. Opin. Cell Biol. 14, 706–714 (2002).

    Article  CAS  Google Scholar 

  4. Shah, J.V. & Cleveland, D.W. Waiting for anaphase: Mad2 and the spindle assembly checkpoint. Cell 103, 997–1000 (2000).

    Article  CAS  Google Scholar 

  5. Tang, Z., Bharadwaj, R., Li, B. & Yu, H. Mad2-independent inhibition of APCCdc20 by the mitotic checkpoint protein BubR1. Dev. Cell 1, 227–237 (2001).

    Article  CAS  Google Scholar 

  6. Sudakin, V., Chan, G.K. & Yen, T.J. Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2. J. Cell Biol. 154, 925–936 (2001).

    Article  CAS  Google Scholar 

  7. Fang, G. Checkpoint protein BubR1 acts synergistically with Mad2 to inhibit anaphase-promoting complex. Mol. Biol. Cell 13, 755–766 (2002).

    Article  CAS  Google Scholar 

  8. Chen, R.H. BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1. J. Cell Biol. 158, 487–496 (2002).

    Article  CAS  Google Scholar 

  9. Millband, D.N. & Hardwick, K.G. Fission yeast Mad3p is required for Mad2p to inhibit the anaphase-promoting complex and localizes to kinetochores in a Bub1p-, Bub3p-, and Mph1p-dependent manner. Mol. Cell. Biol. 22, 2728–2742 (2002).

    Article  CAS  Google Scholar 

  10. Chen, R.H., Brady, D.M., Smith, D., Murray, A.W. & Hardwick, K.G. The spindle checkpoint of budding yeast depends on a tight complex between the Mad1 and Mad2 proteins. Mol. Biol. Cell 10, 2607–2618 (1999).

    Article  CAS  Google Scholar 

  11. Luo, X., Tang, Z., Rizo, J. & Yu, H. The Mad2 spindle checkpoint protein undergoes similar major conformational changes upon binding to either Mad1 or Cdc20. Mol. Cell 9, 59–71 (2002).

    Article  Google Scholar 

  12. Chung, E. & Chen, R.H. Spindle checkpoint requires Mad1-bound and Mad1-free Mad2. Mol. Biol. Cell 13, 1501–1511 (2002).

    Article  CAS  Google Scholar 

  13. Habu, T., Kim, S.H., Weinstein, J. & Matsumoto, T. Identification of a MAD2-binding protein, CMT2, and its role in mitosis. EMBO J. 21, 6419–6428 (2002).

    Article  CAS  Google Scholar 

  14. Wassmann, K., Liberal, V. & Benezra, R. Mad2 phosphorylation regulates its association with Mad1 and the APC/C. EMBO J. 22, 797–806 (2003).

    Article  CAS  Google Scholar 

  15. Sironi, L. et al. Crystal structure of the tetrameric Mad1-Mad2 core complex: implications of a 'safety belt' binding mechanism for the spindle checkpoint. EMBO J. 21, 2496–2506 (2002).

    Article  CAS  Google Scholar 

  16. Luo, X. et al. Structure of the Mad2 spindle assembly checkpoint protein and its interaction with Cdc20. Nat. Struct. Biol. 7, 224–229 (2000).

    Article  CAS  Google Scholar 

  17. Musacchio, A. & Hardwick, K.G. The spindle checkpoint: structural insights into dynamic signaling. Nat. Rev. Mol. Cell Biol. 3, 731–741 (2002).

    Article  CAS  Google Scholar 

  18. Sironi, L. et al. Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is required for the spindle checkpoint. EMBO J. 20, 6371–6382 (2001).

    Article  CAS  Google Scholar 

  19. Fang, G., Yu, H. & Kirschner, M.W. The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation. Genes Dev. 12, 1871–1883 (1998).

    Article  CAS  Google Scholar 

  20. Canman, J.C., Salmon, E.D. & Fang, G. Inducing precocious anaphase in cultured mammalian cells. Cell Motil. Cytoskeleton 52, 61–65 (2002).

    Article  Google Scholar 

  21. Altmann, F., Staudacher, E., Wilson, I.B. & Marz, L. Insect cells as hosts for the expression of recombinant glycoproteins. Glycoconj. J. 16, 109–123 (1999).

    Article  CAS  Google Scholar 

  22. Dhalluin, C. et al. Structural basis of SNT PTB domain interactions with distinct neurotrophic receptors. Mol. Cell 6, 921–929 (2000).

    Article  CAS  Google Scholar 

  23. Bullough, P.A., Hughson, F.M., Skehel, J.J. & Wiley, D.C. Structure of influenza haemagglutinin at the pH of membrane fusion. Nature 371, 37–43 (1994).

    Article  CAS  Google Scholar 

  24. Sauter, N.K., Mau, T., Rader, S.D. & Agard, D.A. Structure of α-lytic protease complexed with its pro region. Nat. Struct. Biol. 5, 945–950 (1998).

    Article  CAS  Google Scholar 

  25. Barrientos, L.G. et al. The domain-swapped dimer of cyanovirin-N is in a metastable folded state: reconciliation of X-ray and NMR structures. Structure 10, 673–686 (2002).

    Article  CAS  Google Scholar 

  26. Khazanovich, N., Bateman, K., Chernaia, M., Michalak, M. & James, M. Crystal structure of the yeast cell-cycle control protein, p13suc1, in a strand-exchanged dimer. Structure 4, 299–309 (1996).

    Article  CAS  Google Scholar 

  27. Ye, S. & Goldsmith, E.J. Serpins and other covalent protease inhibitors. Curr. Opin. Struct. Biol. 11, 740–745 (2001).

    Article  CAS  Google Scholar 

  28. Volkman, B.F., Lipson, D., Wemmer, D.E. & Kern, D. Two-state allosteric behavior in a single-domain signaling protein. Science 291, 2429–2433 (2001).

    Article  CAS  Google Scholar 

  29. James, L.C., Roversi, P. & Tawfik, D.S. Antibody multispecificity mediated by conformational diversity. Science 299, 1362–1367 (2003).

    Article  CAS  Google Scholar 

  30. Prusiner, S.B. Prions. Proc. Natl. Acad. Sci. USA 95, 13363–13383 (1998).

    Article  CAS  Google Scholar 

  31. Mottonen, J. et al. Structural basis of latency in plasminogen activator inhibitor-1. Nature 355, 270–273 (1992).

    Article  CAS  Google Scholar 

  32. Howell, B.J., Hoffman, D.B., Fang, G., Murray, A.W. & Salmon, E.D. Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells. J. Cell Biol. 150, 1233–1250 (2000).

    Article  CAS  Google Scholar 

  33. Hardwick, K.G., Weiss, E., Luca, F.C., Winey, M. & Murray, A.W. Activation of the budding yeast spindle assembly checkpoint without mitotic spindle disruption. Science 273, 953–956 (1996).

    Article  CAS  Google Scholar 

  34. Seeley, T.W., Wang, L. & Zhen, J.Y. Phosphorylation of human MAD1 by the BUB1 kinase in vitro . Biochem. Biophys. Res. Commun. 257, 589–595 (1999).

    Article  CAS  Google Scholar 

  35. Clore, G.M. & Gronenborn, A.M. NMR structure determination of proteins and protein complexes larger than 20 kDa. Curr. Opin. Chem. Biol. 2, 564–570 (1998).

    Article  CAS  Google Scholar 

  36. Gardner, K.H. & Kay, L.E. The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins. Annu. Rev. Biophys. Biomol. Struct. 27, 357–406 (1998).

    Article  CAS  Google Scholar 

  37. Cornilescu, G., Delaglio, F. & Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999).

    Article  CAS  Google Scholar 

  38. Brünger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    Article  Google Scholar 

  39. Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  Google Scholar 

  40. Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

  41. Merritt, E.A. & Bacon, D.J. Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997).

    Article  CAS  Google Scholar 

  42. Kuzmic, P. Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV Proteinase. Anal. Biochem. 237, 260–273 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Y. Liu for reading the manuscript critically and R. Hampton for suggestions. This work was supported by the American Cancer Society (to X.L.), Association pour la Recherche contre le Cancer (ARC) (to K.W.), the Ministry of Education, Culture, Sports, Science and Technology of Japan (COE Research) (to T.M.), the Welch Foundation (to J.R. and H.Y.), the Packard Foundation (to H.Y.), the Burroughs Wellcome Fund (to H.Y.), the W.M. Keck Foundation (to H.Y.) and the US National Institutes of Health (to H.Y.).

Author information

Author notes

  1. Xuelian Luo, Zhanyun Tang and Guohong Xia: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, 75390, Texas, USA

    Xuelian Luo, Zhanyun Tang, Guohong Xia, Josep Rizo & Hongtao Yu

  2. Department of Biochemistry, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, 75390, Texas, USA

    Xuelian Luo & Josep Rizo

  3. Laboratoire de Biologie Cellulaire du Développement, UMR7622, CNRS, Université Pierre et Marie Curie, 9 Quai Saint Bernard, Paris, 75005, France

    Katja Wassmann

  4. Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, Japan

    Tomohiro Matsumoto

Authors
  1. Xuelian Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhanyun Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Guohong Xia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Katja Wassmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tomohiro Matsumoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Josep Rizo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hongtao Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Josep Rizo or Hongtao Yu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

The HSQC spectrum of Mad2–MBP1 is more similar to N2-Mad2 R133A than to N1-Mad2 R133A.(a) The sum of 1H and 15N chemical shift differences (Min Δδ=(1HΔδ2 +( 15NΔδ*0.17)2)½)between N1-Mad2 R133A and Mad2–MBP1 is plotted against residue numbers with the positions of the secondary structural elements of N1-Mad2 indicated above.(b)The sum of 1H and 15 N chemical shift differences between N2-Mad2 R133A and Mad2–MBP1 is plotted against residue numbers.The residues with the largest chemical shift perturbations are involved in MBP1 binding. (JPG 90 kb)

Supplementary Fig. 2

Mad2 R133A refolds into both N1 and N2 forms with a 1:2 ratio.(a) Overlay of the 1H- 15N HSQC spectra of 15N-labeled N2-Mad2 R133A (in black)and the refolded Mad2 R133A (in red).The majority of Mad2 R133A refolds into the N2 conformer.However,there is an additional set of peaks with weaker intensity that belongs to the N1 form.(b) Expansion of a well-resolved region of the HSQC spectra shown in a (boxed)with several cross- peaks labeled.The cross-peaks belonging to the N2 conformer have roughly twice the intensity of the corresponding signals of the N1 conformer. (JPG 104 kb)

Supplementary Fig. 3

Stereoview of the overlaid backbone traces of the 20 final NMR structures of N2-Mad2 R133A.The β-strands are shown in blue; α-helices in green;loops in gray. (JPG 95 kb)

Supplementary Fig. 4

ΔC-Mad2 and ΔC-Mad2 R133A do not interact with Mad1 or Cdc20 in HeLa cells.(a) HeLa cells were transfected with pCS2-GFP alone or together with the indicated Myc6-tagged Mad2-encoding plasmids.The total cell lysates were blotted with either anti-Mad2 (upper panel)or anti-Myc (lower panel)antibodies.The wild-type Mad2 and Mad2 R133A proteins were expressed at similar levels (about the same concentration as the endogenous Mad2)while the concentrations of ΔC-Mad2 and ΔC-Mad2 R133A were much higher (about 8–10 fold of the endogenous Mad2).(b) HeLa cell lysates were cleared by centrifugation and subjected to immunoprecipitation by anti-Myc or anti-Cdc20 antibody beads. The anti-Myc or anti-Cdc20 immunoprecipitates were then blotted with the indicated antibodies. Both wild-type Mad2 and Mad2 R133A associated with the endogenous Cdc20 and Mad1 proteins in HeLa cells treated with thymidine or nocodazole.Nocodazole-treatment did not further enhance the interactions between Cdc20 and the overexpressed Mad2 proteins because the majority of the cells overexpressing wild-type Mad2 or Mad2 R133A were already arrested at metaphase at the time of addition of thymidine.Despite being expressed at much higher levels as compared to wild-type Mad2 and Mad2 R133A,ΔC-Mad2 and ΔC-Mad2 R133A failed to bind to the endogenous Mad1 or Cdc20 proteins. (JPG 100 kb)

Supplementary Fig. 5

Mad2 dissociated from the Mad1–Mad2 complex adopts the N2 conformation.The Mad1–Mad2 complex was fractionated on a Superdex-200 column followed by a Mono-Q column.This procedure produced enough Mad2 that was not no longer bound to Mad1.Overlay of the 1H- 15N HSQC spectra of 15N-labeled N2-Mad2 R133A (in black)and the Mad2 protein dissociated from a Mad1 fragment obtained with gel filtration chromatography (in red).Based on the patterns of the HSQC spectra,the majority of Mad2 dissociated from Mad1 adopts the N2 conformation. (JPG 124 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Luo, X., Tang, Z., Xia, G. et al. The Mad2 spindle checkpoint protein has two distinct natively folded states. Nat Struct Mol Biol 11, 338–345 (2004). https://doi.org/10.1038/nsmb748

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb748

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing